TSG-6 Is Weakly Chondroprotective in Murine OA but Does not Account for FGF2-Mediated Joint Protection.

OBJECTIVE: Tumor necrosis factor α-stimulated gene 6 (TSG-6) is an anti-inflammatory protein highly expressed in osteoarthritis (OA), but its influence on the course of OA is unknown. METHODS: Cartilage injury was assessed by murine hip avulsion or by recutting rested explants. Forty-two previously validated injury genes were quantified by real-time polymerase chain reaction in whole joints following destabilization of the medial meniscus (DMM) (6 hours and 7 days). Joint pathology was assessed at 8 and 12 weeks following DMM in 10-week-old male and female fibroblast growth factor 2 (FGF2)-/- , TSG-6-/- , TSG-6tg (overexpressing), FGF2-/- ;TSG-6tg (8 weeks only) mice, as well as strain-matched, wild-type controls. In vivo cartilage repair was assessed 8 weeks following focal cartilage injury in TSG-6tg and control mice. FGF2 release following cartilage injury was measured by enzyme-linked immunosorbent assay. RESULTS: TSG-6 messenger RNA upregulation was strongly FGF2-dependent upon injury in vitro and in vivo. Fifteeen inflammatory genes were significantly increased in TSG-6-/- joints, including IL1α, Ccl2, and Adamts5 compared with wild type. Six genes were significantly suppressed in TSG-6-/- joints including Timp1, Inhibin βA, and podoplanin (known FGF2 target genes). FGF2 release upon cartilage injury was not influenced by levels of TSG-6. Cartilage degradation was significantly increased at 12 weeks post-DMM in male TSG-6-/- mice, with a nonsignificant 30% reduction in disease seen in TSG-6tg mice. No differences were observed in cartilage repair between genotypes. TSG-6 overexpression was unable to prevent accelerated OA in FGF2-/- mice. CONCLUSION: TSG-6 influences early gene regulation in the destabilized joint and exerts a modest late chondroprotective effect. Although strongly FGF2 dependent, TSG-6 does not explain the strong chondroprotective effect of FGF2

In a surgical model of OA female mice compared to males display pain at an early stage of disease and differential regulation of pain sensitizers

Meeting Abstract from British Society for Matrix Biology Autumn 2020 Meeting

The ciliary protein intraflagellar transport 88 is required for the maturation, homeostasis and mechanoadaptation of articular cartilage

Purpose: The development, maturation and maintenance of articular cartilage depends on the integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling. Aberrant mechanics and the post-natal activation of Hh signalling have both been associated with the development of murine and human OA. It remains to be fully elucidated how chondrocytes transduce and integrate these cues in vivo. Chondrocytes assemble a primary cilium, a microtubule-based organelle with a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate ciliary IFT helps tune the chondrocyte response to Hh ligand and the anabolic matrix response to cell compression. Recently, we have shown loss of function of IFT88, inhibiting ciliogenesis in chondrocytes, impairs LRP-1 mediated endocytotic clearance of proteases, resulting in increased aggrecanolysis. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown.Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;IFT88fl/fl : cKO hereafter). Cre activity was validated by qPCR, and using a ROSA26tdtomato reporter line. IFT88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, and OARSI score and by means of immunohistochemistry (IHC). The DMM model, which destabilising the joint, was performed at 10 weeks of age. Means ± S.D are quoted throughout, Mann-Whitney U-tests used for statistical comparisons.Results: The Tdtomato reporter demonstrated ACANcre activity in hip and throughout knee cartilage. Tamoxifen treatment of cKO mice resulted in a 50% reduction of IFT88 mRNA in articular cartilage (p=0.02, n=6 ctrl, 14 cKO). In control mice, tibial articular cartilage thickens between 8 and 22 weeks of age, most notably on the medial plateau. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age. IFT88 depletion (cKO) resulted in thinner medial articular cartilage (MC), compared with controls, at all 5 time-points. Fig 1.A shows MC thickness in adult control (crosses) and cKO (circles). In control mice, MC thickness increased from 99.0 +/- 9.2 μm at 8 weeks of age to 108.9 +/- 7.2 μm at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.2 +/- 7.4 μm, p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MC thickness in cKO was 90.2 +/- 3.3 μm compared with 111.6 +/- 10.1 μm in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MC had continued to thin, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score was unaffected. At all time-points thinning was attributable to loss of calcified cartilage. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope staining or the expression of LRP-1β. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO mice.Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued adaptation of adolescent and adult articular cartilage, in the medial loading environment. IFT88 deletion inhibits MC thickening, leading to atrophy, which then predisposes the joint to spontaneous OA as the mouse ages. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of IFT88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of intrinsic cartilage Hh signalling. Mechanistic experiments, dissecting the relative roles and integration of IFT, mechanics and Hh in the context of adult cartilage are on-going. We conclude that IFT88 maintains a profound influence in post-natal articular cartilage homeostasis and protection from OA.</div

Studying osteoarthritis pathogenesis in mice

With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome-wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development. © 2018 by John Wiley &amp; Sons, Inc

Studying osteoarthritis pathogenesis in mice

With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome-wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development. © 2018 by John Wiley and Sons, Inc